The present invention generally relates to edible vaccines produced in plants, useful for the treatment of autoimmune disease.
Systemic immunosuppressive therapy in autoimmune disease and transplantation is associated with increased rates of infection, malignancy and numerous side effects. The induction of antigen-specific hyporesponsiveness without drugs is therefore desirable. Immune responses to orally administered proteins is intrinsically modulated and may induce a state of systemic hyporesponsiveness termed oral tolerance (Kay et al., (1989), Immunology, vol. 66, pp. 416-421; Peng et al., (1990), Clin. exp. Immunol., vol. 81, pp. 510-515; Lamont et al., (1989), Immunology, vol. 66, pp. 595-599). Although many factors have been implicated in this phenomenon, including soluble mediators and suppressor T cells, it is apparent that antigen processing by mucosal tissue is critical for this effect.
Various studies have reported oral administration of antigens, thought to be associated with autoimmune diseases, in an effort to induce oral tolerance and prevent or reduce autoimmune disease.
In International Patent Application Publication No. WO 92/07581, and in Weiner et al., (1992), Proc. Natl. Acad. Sci. USA, vol. 89, pp. 7762-7766, Wiener et al. describe suppression of the mammalian response to allografts by oral administration of splenocytes or splenocyte preparations from tissue donors, or oral administration of short synthesized peptides corresponding to fragments of class II Major Histocompatibility Complex (MHC) proteins.
There are, however, several problems associated with the approach of oral tolerance. First, the complexity of foreign peptide presentation in transplantation makes it difficult to identify peptide sequences suitable for induction of tolerance.
Second, the induction of oral tolerance to antigens is dose dependent and an insufficient level of an oral antigen may prime gut lymphocytes and cause the opposite and undesired effect of sensitization. It is therefore necessary to be able to obtain and deliver the antigens in a sufficient quantity to induce oral tolerance.
Third, the nature of the peptide itself may stimulate rather than reduce immune responsiveness.
If the entire amino acid sequence of an antigen protein is used to induce oral tolerance, then a greater array of potentially tolerance-inducing peptides will be presented to the immune system. If complex antigens such as MHC proteins or other transplantation antigens are to be used as intact proteins, however, it is difficult to obtain these proteins in sufficient quantities by in vitro synthesis. It may also be difficult to deliver a sufficient quantity of these protein antigens to induce oral tolerance.
Transgenic plants have been used to express a variety of single chain heterologous polypeptides with considerable success (Trudel et al., (1992), Plant Science, v. 87, pp. 55-67). More complex multi-chain proteins such as antibodies have also been expressed in plants but with less consistent results (Swain, W. F. (1991), Tibtech, v. 9. p. 107).
It has been proposed that viral antigens expressed in plants may provide an xe2x80x9cedible vaccinexe2x80x9d, whereby ingestion of plants containing the viral antigen by a human would stimulate an increased immune response and provide immunization against the virus (Mason et al., (1992), Proc. Natl. Acad. Sci. USA, vol. 89, pp. 11745-11749).
The high cost of production and purification of synthetic peptides manufactured by chemical or fermentation based processes may prevent their broad scale use as oral vaccines. The production of immunogenic proteins in transgenic plants, on the other hand, offers an economical alternative. Attempts have been made to produce transgenic plants that express bacterial antigens of E. coli and Streptococcus mutants. For instance, Curtiss et al. (WO 90/0248) report the transformation of sunflower with the E. coli LT-B gene. Also, the expression of LT-B and its assembly into GM1-binding pentamers in tobacco and potato plants has been reported (Haq et al. 1995). Additionally, Arntzen et al. (WO 96/12801) disclose vectors for the independent and coordinate expression of LT-A and LT-B, which optionally contain a SEKDEL microsomal retention signal. The transformation of tobacco and potato plants with these genes is also described.
It has been shown that the inclusion of KDEL amino acid sequences at the carboxy terminus of a protein can enhance the recognition for that protein by the plant ER retention machinery (see, e.g., Munro and Pelham 1987). However, such modifications can be problematic because other factors, such as protein conformation or protein folding in the transformed cells, may interfere with the accessability of this carboxy terminal signal to the plant ER retention machinery. Retention of key biological properties in the recombinant proteins produced in plants, specifically ligand binding and the presentation of antigenic epitopes, is of considerable importance to the successful production of edible vaccines in transgenic plants.
Oral vaccines derived from transgenic plants are potentially an effective and inexpensive means for inducing oral tolerance, and secretory immune responses to enterotoxins, in mammals including humans. Recently, plants have been used for the production of vaccine antigens such as viral capsid proteins and bacterial enterotoxins (Haq, T. A., Mason, H. S., Clements, J. D., and Arntzen, C. J., 1995, Science 268:714-716; Mason, H. S., Ball, J. M., Shi, J. -J., Jiang, X., Estes, M. K., and Arntzen, C. J. 1996, Proc. Natl. Acad. Sci. USA 93:5335-5340; Arakawa, T., Chong, D. K. X., and Langridge, W. H. R. 1998, Nat. Biotechnol. 16:292-297; Arakawa, T., Chong, D. K. X., Merritt, J. L., and Langridge, W. H. R. 1997, Transgenic Res. 6:403-413). Production of autoantigens in food plants for the induction of oral tolerance offers the following advantages. Plants can synthesize proteins at low cost and protein intake can be achieved through direct consumption of the edible plant tissues.
Cholera toxin (CT) is a potent mucosal immunogen that has strong mucosal adjuvant qualities (Clements et al., 1988; Holmgren et al., 1993). Thus, immune responses against other antigens can be enhanced by co-presentation with low doses of CT.
The nontoxic cholera toxin B subunit (CTB) has been used to increase the tolerogenic nature of orally administered antigens based on the affinity of CTB for GM1-ganglioside, a cell surface receptor located on the M cells in gut-associated lymphoid tissues (GALT) and enterocytes in the intestinal villi (Sun, J. -B., Holmgren, J., and Czerkinsky, C., 1994, Proc. Natl. Acad. Sci. USA 91:10795-10799; Weiner, H. L., 1994, Proc. Natl. Acad. Sci. USA 91:10762-10765). This application of CTB has proven useful in the prevention and treatment of autoimmune diseases in animals (Sun, J. -B., Rask, C., Olsson, T., Holmgren, J., and Czerkinsky, C., 1996, Proc. Natl. Acad. Sci. USA 93:7196-7201; Bergerot, I., Ploix, C., Petersen, J., Moulin, V., Rask, C., Fabien, N., Lindblad, M., Mayer, A., Czerkinsky, C., Holmgren, J., and Thivolet, C. 1997, Proc. Natl. Acad. Sci. USA 94:4610-4614).
There is a need in the art for a vaccine that is capable of delivering the entire amino acid sequence of a complex autoantigen for the induction of oral tolerance.
There is also a need in the art for a vaccine that is capable of delivering sufficient quantities of the entire amino acid sequence of a complex autoantigen for the induction of oral tolerance.
There is also a need in the art for an economical method for producing safe vaccines.
There is also a need in the art for an economical method for producing safe, edible vaccines.
There is also a need in the art for an economical method for producing safe, edible vaccines in plants.
There is also a need in the art for an edible vaccine that can facilitate efficient, site-specific delivery of a concentration of an autoantigen sufficient to induce oral tolerance.
There is also a need in the art for an edible vaccine that can facilitate efficient, site-specific delivery of a concentration of an autoantigen sufficient to induce oral tolerance, that is produced in plants.
There is also a need in the art for an edible vaccine that is useful for the induction of oral tolerance and the treatment of autoimmune disease.
The present invention provides chimeric gene constructs comprising a CTB coding sequence and an autoantigen coding sequence, plant cells and transgenic plants transformed with said chimeric gene constructs, and methods of preparing an edible vaccine from these plant cells and transgenic plants. The present invention also provides methods of treating autoimmune disease with edible vaccines, compositions comprising edible vaccines according to the invention and fusion proteins comprising a CTB-autoantigen protein.
The invention encompasses a chimeric gene construct comprising: a plant promoter operatively associated with DNA comprising a CTB coding sequence and an autoantigen coding sequence.
In preferred embodiments, the chimeric gene construct may further comprise a translation enhancer operatively associated with the autoantigen coding sequence wherein the translation enhancer is fused between the CTB coding sequence and the autoantigen coding sequence.
In other preferred embodiments, the translational enhancer is fused between the CTB coding sequence and the autoantigen coding sequence.
In other preferred embodiments, the construct may further comprise a transcriptional enhancer operatively associated with the translational enhancer and the autoantigen coding sequence; wherein the transcriptional enhancer is fused to the translation enhancer and the autoantigen coding sequence.
Preferably, the transcriptional enhancer is fused to the translation enhancer and the autoantigen coding sequence.
As used herein, xe2x80x9coperatively associatedxe2x80x9d refers to a cis genetic linkage which permits functional association of a genetic element and one or more coding sequences, whether the genetic element is a promoter or an enhancer (transcriptional or translational).
xe2x80x9cFused toxe2x80x9d refers to a cis genetic linkage; if translation is involved, xe2x80x9cfused toxe2x80x9d permits a fusion protein to be produced.
In other embodiments, the chimeric gene construct encodes for a protein antigenically related to a corresponding authentic autoantigen.
In other preferred embodiments, the chimeric gene construct may further comprise one or more internal ribosome entry site elements and one or more additional antigen coding sequences, wherein the one or more entry site elements are operatively associated with the one or more additional antigen coding sequences so as to permit their translation.
In other preferred embodiments, the chimeric gene construct may further comprise a nucleotide sequence encoding a microsomal retention signal wherein the retention signal is fused to the 3xe2x80x2 end of the autoantigen coding sequence, and; a nucleotide sequence encoding a flexible hinge peptide wherein the flexible hinge peptide is fused to the transcriptional enhancer and the autoantigen coding sequence.
The invention also encompasses a plant cell transformed with the chimeric gene construct described herein conferring production of the encoded protein in the plant cell.
The invention also encompasses a transgenic plant transformed with the chimeric gene construct according to the present invention conferring production of the encoded protein in the transgenic plant.
The invention also encompasses a method for preparing an autoantigen in a plant cell comprising growing plant cells transformed with the chimeric gene construct according to the present invention; and expressing the autoantigen coding sequence an the plant cells to confer production of the encoded protein in the plant cells.
The invention also encompasses a method for preparing an autoantigen in a transgenic plant comprising transforming a transgenic plant with the chimeric gene construct according to the present invention; and expressing the autoantigen coding sequence in the transgenic plant to confer production of the encoded protein in the transgenic plant.
The invention also encompasses a composition comprising plant cells transformed with the chimeric gene construct described herein in admixture with a physiologically compatible carrier.
The invention also encompasses a composition comprising a transgenic plant transformed with the chimeric gene construct according to the present invention in admixture with a physiologically compatible carrier.
The invention also encompasses a method of treating an autoimmune disease, comprising: administering a composition described herein to a mammal suspected of suffering from the autoimmune disease, in an amount sufficient to ameliorate symptoms or to prevent the disease.
The invention also encompasses a method of preventing or treating a T-cell mediated autoimmune disease, comprising administering a composition as described herein to a mammal suspected of suffering from the T-cell mediated autoimmune disease, in an amount sufficient to ameliorate symptoms of or to prevent the disease.
The invention also encompasses a method of preventing or treating an autoimmune disease, comprising administering a composition as described herein to a mammal suspected of suffering from the autoimmune disease, in an amount sufficient to ameliorate symptoms of or to prevent the disease.
The invention also encompasses a method of treating an T-cell mediated autoimmune disease, comprising administering a composition described herein to a mammal suspected of suffering from the T-cell mediated autoimmune disease, in an amount sufficient to ameliorate symptoms of or to prevent the disease.
The invention also encompasses a kit comprising an autoimmune disease agent comprising a composition as described herein and packaging therefore.
The invention also encompasses a kit comprising a T-cell mediated autoimmune disease agent comprising a composition described herein and packaging therefore.
The invention also encompasses a kit comprising an autoimmune disease agent comprising a composition as described herein and packaging therefore.
The invention also encompasses a kit comprising a T-cell mediated autoimmune disease agent comprising a composition as described herein and packaging therefore.
The invention also encompasses an edible composition comprising edible plant cells transformed with the chimeric construct described herein admixed with a physiologically compatible carrier.
The invention also encompasses an edible composition comprising an edible transgenic plant transformed with the chimeric construct described herein admixed with a physiologically compatible carrier.
The invention also encompasses a vector comprising the chimeric gene construct described herein.
Preferably, in the vector containing the chimeric gene construct described herein, the autoantigen is a B-cell autoantigen, which may be glutamic acid decarboxylase or insulin.
The invention also encompasses a fusion protein comprising a sufficient amount of amino acid sequence of a CTB protein such that the fusion protein is expressed and is capable of forming a pentameric structure as determined by GM1-ganglioside binding and an amino acid sequence of an autoantigen.
Preferably, the fusion protein further comprise a microsomal retention signal and a flexible hinge peptide.
As used herein, xe2x80x9ctranscriptional enhancerxe2x80x9d refers to a regulatory DNA sequence to which gene regulatory proteins bind, that influences the rate of transcription of a structural gene by facilitating transcription initiation thereby increasing the amount of messenger RNA that is transcribed from a gene. By increasing messenger RNA levels, a transcriptional enhancer ultimately increases the level of the protein product that is produced from the corresponding messenger RNA molecule by at least 10 fold, and preferably 10-100 fold. a transcriptional enhancer can be 50 bp to 10,000 base pairs and preferably 50-150 base pairs, can be located upstream or downstream of a gene or within the coding sequence of a gene and can function in either a 3xe2x80x2-5xe2x80x2 or a 5xe2x80x2-3xe2x80x2 orientation.
In a particularly preferred embodiment of the invention, the octopine synthase (OCS) enhancer element is particularly useful as a transcriptional enhancer. According to the invention, the OCS element is used to increase the amount of protein produced from the mas P1 promoter and the mas P2 promoter. The mas P1 and P2 promoters contain one and two OCS elements respectively. Additional enhancers useful according to the invention are provided in the section entitled xe2x80x9cProduction of an Edible Vaccinexe2x80x9d.
As used herein, xe2x80x9ctranslation enhancerxe2x80x9d refers to a regulatory element that enhances translation by at least 5-fold, preferably 5-fold to 100-fold and more preferably from 8-fold to 21-fold.
Translation enhancers that are useful according to the invention are preferably isolated from plant viruses and include but are not limited to translation enhancers isolated from the 5xe2x80x2 untranslated leader elements from tobacco etch virus (an 80 bp element), tobacco mosaic virus, cucumber mosaic virus and alfalfa mosaic virus.
By xe2x80x9cprotein that is antigenically relatedxe2x80x9d is meant a protein that has an amino acid sequence that is 60-80% identical, and is immunologically cross reactive as determined by immunoblotting, immunoprecipitation or ELISA. For example, CTB and LTB are antigenically related.
By xe2x80x9cauthentic autoantigenxe2x80x9d is meant the native form of the autoantigen.
By xe2x80x9cautoantigen production in transgenic plantxe2x80x9d is meant translation or synthesis of a CTB fusion protein according to the invention, in tuber tissues in an amount that can be detected by the methods of immunoblotting, immunoprecipitation or ELISA. Preferably the amount of CTB-fusion protein that is produced in a transgenic plant is 0.1% to 1% of the total soluble tuber protein, and preferably 0.3-0.6%.
By xe2x80x9cedible vaccinexe2x80x9d is meant a food plant delivering an autoantigen which is protective against an infectious disease or an autoimmune disease. In particular, the invention provides for an edible vaccine that induces a state of immunological tolerance.
By xe2x80x9cinfectious diseasexe2x80x9d is meant a disease caused by an infectious agent.
The invention provides for the expression of CTB and appropriate mammalian self-antigens, for example transplantation antigens or autoantigens, in plants and the administration of these plants or plant materials derived from these plants to a mammal to produce oral tolerance to the expressed mammalian self-antigens in order to control or suppress allograft rejection or autoimmune responses in the mammal.
Further features and advantages of the invention are as follows. The present invention is a highly effective vaccine against autoimmune disease that overcomes certain deficiencies of vaccines prepared to date. In particular, the vaccine of the claimed invention is an edible vaccine that can be produced via a low cost method in plants. The edible vaccine according to the invention also offers the advantages of delivering high concentrations of an autoantigen in a site-specific manner.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof.